LETTERS TO THE EDITOR
sensitivity analyses might help decrease the risk of type I error. Such analyses might include nonparametric correlation tests (Spearman’s r), delimiting correlation tests to the schizophrenia subjects with lower Zif268 mRNA expression (N549/62), reincluding outliers, and performing adjusted linear regressions. In addition, providing more detail on how included subjects were determined (e.g., consecutive/unselected versus convenience/ selected, proportion of excluded subjects with reasons) would allow an informed assessment of selection bias risk. The authors acknowledged that factors other than decreased Zif268 mRNA expression might also contribute to decreased cortical GAD67 mRNA expression in schizophrenia and discussed several alternative putative mechanisms. However, these alternatives did not include neuroinflammation. Neuropathological and neuroimaging studies have provided evidence implicating neuroinflammatory mechanisms in decreased cortical GAD67 mRNA expression in schizophrenia (2). For example, one neuropathological study documented lower cortical GAD67 mRNA expression among schizophrenia subjects with high versus low proinflammatory cytokine mRNA expression (3). The neuroinflammation model hypothesizes that prenatal insult induces microglial priming, particularly within white matter, resulting in neuroinflammation-mediated disruption of neuronal migration from periventricular germinal matrix to cortex. Thus, the neuroinflammation model maintains external validity not only with decreased cortical GAD67 mRNA expression findings but also with evidence of increased white matter neuronal density and with structural and functional white matter disconnectivity in schizophrenia, reproducible abnormalities central to neurodevelopmental and neurodegenerative hypotheses. Accordingly, neuroinflammatory mechanisms represent relevant context to the study findings. References 1. Kimoto S, Bazmi HH, Lewis DA: Lower expression of glutamic acid decarboxylase 67 in the prefrontal cortex in schizophrenia: contribution of altered regulation by Zif268. Am J Psychiatry (Epub ahead of print, May 30, 2014) 2. Najjar S, Pearlman DM: Neuroinflammation and white matter pathology in schizophrenia: systematic review. Schizophr Res (Epub ahead of print, June 16, 2014) 3. Fillman SG, Cloonan N, Catts VS, Miller LC, Wong J, McCrossin T, Cairns M, Weickert CS: Increased inflammatory markers identified in the dorsolateral prefrontal cortex of individuals with schizophrenia. Mol Psychiatry 2013; 18:206–214
DANIEL M. PEARLMAN, M.P.H. SOUHEL NAJJAR, M.D.
From the Department of Neurology, New York University School of Medicine, New York; and The Dartmouth Institute for Health Policy and Clinical Practice, Audrey and Theodor Geisel School of Medicine at Dartmouth, Hanover, N.H. The authors report no financial relationships with commercial interests. This letter (doi: 10.1176/appi.ajp.2014.14060701) was accepted for publication in July 2014.
Response to Pearlman and Najjar To the Editor: We thank Drs. Daniel M. Pearlman and Souhel Najjar for their interest in our study. They suggested
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some additional analyses relevant to the relationship between Zif268 and GAD67 mRNA levels in the dorsolateral prefrontal cortex of subjects with schizophrenia, which we provide below. First, the significant positive association we reported between Zif268 and GAD67 mRNA levels in schizophrenia subjects by Pearson’s correlation (r50.29, p50.027) is supported by the requested Spearman’s correlation (r50.33, p50.013) in the same schizophrenia subjects. Second, Pearlman and Najjar suggest looking for the association between Zif268 and GAD67 mRNA levels just within the schizophrenia subjects who had Zif268 mRNA levels lower than their matched comparison subject. We do not think such an analysis is informative because it imputes biological meaning to findings of uncertain significance (e.g., Is a 5% difference within a one subject pair really different from a 5% difference in the opposite direction in another subject pair?). Indeed, as indicated in the Methods section, we used subject pairing to control experimental variance and to reduce biological variance across groups and not a true paired design in which the only difference between subjects in a pair is the independent measure of interest. Thus, the arbitrary exclusion of some subjects with schizophrenia based on pairwise differences would reduce statistical power without a biological or statistical rationale. However, as we reported in the article, the use of benzodiazepines or sodium valproate at the time of death was associated with lower Zif268 mRNA expression, although those subjects with schizophrenia not taking benzodiazepines or sodium valproate at the time of death still had lower Zif268 mRNA levels. Within those schizophrenia subjects, levels of Zif268 and GAD67 mRNAs were strongly correlated (Pearson’s r50.37, p50.030), suggesting that eliminating other sources of variance makes the potential causal role of Zif268 in regulating GAD67 expression more evident. Third, as described in the article, outliers were determined as follows: within each subject group, subjects with Zif268 mRNA levels more than 3 standard deviations from the group mean were considered outliers. Using this criterion, five (four schizophrenia subjects and one comparison subject) of 124 subjects were identified as outliers for Zif268 mRNA levels. Although the inclusion of such subjects is likely only to increase experimental variance, Zif268 and GAD67 mRNA levels were still significantly correlated (r50.29, p50.023) across all schizophrenia subjects examined (N562). Fourth, the subjects included in this study are from well-characterized cohorts that have been previously used in multiple studies. Selection criteria in creating the cohorts involve the measures of tissue quality described in the Methods section and the criteria (age, postmortem interval, etc.) that are used to identify cases appropriate for brain donation. Our approach to the important design issues for case inclusion in postmortem studies has been previously described (1, 2). Finally, Pearlman and Najjar note that we acknowledged that factors other than Zif268 may influence GAD67 mRNA expression, and they suggest that inflammation might be one such mechanism. We agree that the neuroinflammatory model of schizophrenia pathogenesis is important to consider. For example, interferon-induced transmembrane protein was reported to be higher in the dorsolateral prefrontal cortex in subjects with schizophrenia, and levels of interferon-induced transmembrane protein and GAD67 mRNAs were negatively correlated in these subjects (3). As noted in our article, our goal
Am J Psychiatry 171:10, October 2014
LETTERS TO THE EDITOR
was to explore the proximal molecular mediators that could contribute to lower GAD67 mRNA expression in schizophrenia. Thus, because Zif268 directly binds to the promoter region of the GAD67 gene and is expressed in response to neuronal activation, including activation induced by a number of noxious stimuli, it is possible that neuroinflammation could be upstream of lower Zif268 and/or GAD67 mRNA levels in schizophrenia. References 1. Lewis DA: The human brain revisited: opportunities and challenges in postmortem studies of psychiatric disorders. Neuropsychopharmacology 2002; 26:143–154 2. Beneyto M, Sibille E, Lewis DA: Human postmortem brain research in mental illness syndromes, in Neurobiology of Mental Illness.
Am J Psychiatry 171:10, October 2014
Edited by Charney DS, Nestler EJ. New York, Oxford University Press, 2009, pp 202–214 3. Siegel BI, Sengupta EJ, Edelson JR, Lewis DA, Volk DW: Elevated viral restriction factor levels in cortical blood vessels in schizophrenia. Biol Psychiatry 2014; 76:160–167
SOHEI KIMOTO, M.D., PH.D. DAVID A. LEWIS, M.D.
From the Department of Psychiatry and the Department of Neuroscience, University of Pittsburgh, Pittsburgh. The authors’ disclosures appear with the original article. This reply (doi: 10.1176/appi.ajp.2014.14060701r) was accepted for publication in July 2014.
ajp.psychiatryonline.org
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sensitivity analyses might help decrease the risk of type I error. Such analyses might include nonparametric correlation tests (Spearman’s r), delimiting correlation tests to the schizophrenia subjects with lower Zif268 mRNA expression (N549/62), reincluding outliers, and performing adjusted linear regressions. In addition, providing more detail on how included subjects were determined (e.g., consecutive/unselected versus convenience/ selected, proportion of excluded subjects with reasons) would allow an informed assessment of selection bias risk. The authors acknowledged that factors other than decreased Zif268 mRNA expression might also contribute to decreased cortical GAD67 mRNA expression in schizophrenia and discussed several alternative putative mechanisms. However, these alternatives did not include neuroinflammation. Neuropathological and neuroimaging studies have provided evidence implicating neuroinflammatory mechanisms in decreased cortical GAD67 mRNA expression in schizophrenia (2). For example, one neuropathological study documented lower cortical GAD67 mRNA expression among schizophrenia subjects with high versus low proinflammatory cytokine mRNA expression (3). The neuroinflammation model hypothesizes that prenatal insult induces microglial priming, particularly within white matter, resulting in neuroinflammation-mediated disruption of neuronal migration from periventricular germinal matrix to cortex. Thus, the neuroinflammation model maintains external validity not only with decreased cortical GAD67 mRNA expression findings but also with evidence of increased white matter neuronal density and with structural and functional white matter disconnectivity in schizophrenia, reproducible abnormalities central to neurodevelopmental and neurodegenerative hypotheses. Accordingly, neuroinflammatory mechanisms represent relevant context to the study findings. References 1. Kimoto S, Bazmi HH, Lewis DA: Lower expression of glutamic acid decarboxylase 67 in the prefrontal cortex in schizophrenia: contribution of altered regulation by Zif268. Am J Psychiatry (Epub ahead of print, May 30, 2014) 2. Najjar S, Pearlman DM: Neuroinflammation and white matter pathology in schizophrenia: systematic review. Schizophr Res (Epub ahead of print, June 16, 2014) 3. Fillman SG, Cloonan N, Catts VS, Miller LC, Wong J, McCrossin T, Cairns M, Weickert CS: Increased inflammatory markers identified in the dorsolateral prefrontal cortex of individuals with schizophrenia. Mol Psychiatry 2013; 18:206–214
DANIEL M. PEARLMAN, M.P.H. SOUHEL NAJJAR, M.D.
From the Department of Neurology, New York University School of Medicine, New York; and The Dartmouth Institute for Health Policy and Clinical Practice, Audrey and Theodor Geisel School of Medicine at Dartmouth, Hanover, N.H. The authors report no financial relationships with commercial interests. This letter (doi: 10.1176/appi.ajp.2014.14060701) was accepted for publication in July 2014.
Response to Pearlman and Najjar To the Editor: We thank Drs. Daniel M. Pearlman and Souhel Najjar for their interest in our study. They suggested
1118
ajp.psychiatryonline.org
some additional analyses relevant to the relationship between Zif268 and GAD67 mRNA levels in the dorsolateral prefrontal cortex of subjects with schizophrenia, which we provide below. First, the significant positive association we reported between Zif268 and GAD67 mRNA levels in schizophrenia subjects by Pearson’s correlation (r50.29, p50.027) is supported by the requested Spearman’s correlation (r50.33, p50.013) in the same schizophrenia subjects. Second, Pearlman and Najjar suggest looking for the association between Zif268 and GAD67 mRNA levels just within the schizophrenia subjects who had Zif268 mRNA levels lower than their matched comparison subject. We do not think such an analysis is informative because it imputes biological meaning to findings of uncertain significance (e.g., Is a 5% difference within a one subject pair really different from a 5% difference in the opposite direction in another subject pair?). Indeed, as indicated in the Methods section, we used subject pairing to control experimental variance and to reduce biological variance across groups and not a true paired design in which the only difference between subjects in a pair is the independent measure of interest. Thus, the arbitrary exclusion of some subjects with schizophrenia based on pairwise differences would reduce statistical power without a biological or statistical rationale. However, as we reported in the article, the use of benzodiazepines or sodium valproate at the time of death was associated with lower Zif268 mRNA expression, although those subjects with schizophrenia not taking benzodiazepines or sodium valproate at the time of death still had lower Zif268 mRNA levels. Within those schizophrenia subjects, levels of Zif268 and GAD67 mRNAs were strongly correlated (Pearson’s r50.37, p50.030), suggesting that eliminating other sources of variance makes the potential causal role of Zif268 in regulating GAD67 expression more evident. Third, as described in the article, outliers were determined as follows: within each subject group, subjects with Zif268 mRNA levels more than 3 standard deviations from the group mean were considered outliers. Using this criterion, five (four schizophrenia subjects and one comparison subject) of 124 subjects were identified as outliers for Zif268 mRNA levels. Although the inclusion of such subjects is likely only to increase experimental variance, Zif268 and GAD67 mRNA levels were still significantly correlated (r50.29, p50.023) across all schizophrenia subjects examined (N562). Fourth, the subjects included in this study are from well-characterized cohorts that have been previously used in multiple studies. Selection criteria in creating the cohorts involve the measures of tissue quality described in the Methods section and the criteria (age, postmortem interval, etc.) that are used to identify cases appropriate for brain donation. Our approach to the important design issues for case inclusion in postmortem studies has been previously described (1, 2). Finally, Pearlman and Najjar note that we acknowledged that factors other than Zif268 may influence GAD67 mRNA expression, and they suggest that inflammation might be one such mechanism. We agree that the neuroinflammatory model of schizophrenia pathogenesis is important to consider. For example, interferon-induced transmembrane protein was reported to be higher in the dorsolateral prefrontal cortex in subjects with schizophrenia, and levels of interferon-induced transmembrane protein and GAD67 mRNAs were negatively correlated in these subjects (3). As noted in our article, our goal
Am J Psychiatry 171:10, October 2014
LETTERS TO THE EDITOR
was to explore the proximal molecular mediators that could contribute to lower GAD67 mRNA expression in schizophrenia. Thus, because Zif268 directly binds to the promoter region of the GAD67 gene and is expressed in response to neuronal activation, including activation induced by a number of noxious stimuli, it is possible that neuroinflammation could be upstream of lower Zif268 and/or GAD67 mRNA levels in schizophrenia. References 1. Lewis DA: The human brain revisited: opportunities and challenges in postmortem studies of psychiatric disorders. Neuropsychopharmacology 2002; 26:143–154 2. Beneyto M, Sibille E, Lewis DA: Human postmortem brain research in mental illness syndromes, in Neurobiology of Mental Illness.
Am J Psychiatry 171:10, October 2014
Edited by Charney DS, Nestler EJ. New York, Oxford University Press, 2009, pp 202–214 3. Siegel BI, Sengupta EJ, Edelson JR, Lewis DA, Volk DW: Elevated viral restriction factor levels in cortical blood vessels in schizophrenia. Biol Psychiatry 2014; 76:160–167
SOHEI KIMOTO, M.D., PH.D. DAVID A. LEWIS, M.D.
From the Department of Psychiatry and the Department of Neuroscience, University of Pittsburgh, Pittsburgh. The authors’ disclosures appear with the original article. This reply (doi: 10.1176/appi.ajp.2014.14060701r) was accepted for publication in July 2014.
ajp.psychiatryonline.org
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